Malaria is an acute or chronic disease caused by the presence of sporozoan parasites of the genus Plasmodium in the host's red blood cells (erythrocytes). Four different species can cause the disease in humans: Plasmodium faciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. Malaria merozoites (the vertebrate infective stage of the Plasmodium parasite) must attach to and invade a new erythrocyte in order to continue parasite development in the blood of an infected host. Symptoms of the disease (including shaking, chills, and fever) appear only when erythrocyte cells infected with the parasites rupture to release more parasites into the blood stream.
Plasmodium merozoites control invasion into erythrocytes by a series of molecular interactions. In particular, malaria parasites express Erythrocyte Binding Proteins (EBPs) during the invasive merozoite stage, and those EBPs interact with host cell surface proteins to facilitate host cell recognition and invasion. Determinants on human erythrocytes are essential for invasion of human erythrocytes by Plasmodium and much of current knowledge about malaria merozoite invasion of erythrocytes is based on observations of the primate malaria parasite Plasmodium knowlesi [Dvorak et al., Science 187: 748-750 (1975)]. Plasmodium knowlesi and Plasmodium vivax both require the presence of the Duffy blood group antigen in order to invade human erythrocytes [Miller et al., Science 189: 561-563 (1975) and Miller et al., N. Engl. J. Med. 295: 302-304 (1976)]; however, Plasmodium falciparum usually requires sialic acid-dependent epitopes on glycophorin A in order to invade human erythrocytes [Camus et al., Science 230, 553-6, 1985].
Merozoites interact with the erythrocyte by initially attaching at the point of first contact and then undergo apical reorientation. A junction then forms between the apical end of the merozoite and the erythrocyte, followed by vacuole formation and entry of the merozoite into the vacuole [Aikawa et al., J. Cell Biol. 77, 72-82 (1978)]. Junction formation and merozoite entry into the erythrocyte do not occur on erythrocytes lacking the appropriate receptor [Miller et al., J. Exp. Med. 149, 172-184 (1979)], indicating that a specific receptor is involved in apical junction formation but not initial attachment. Junction formation is the critical point of the invasion process when a parasite must commit itself to invade a particular cell, since subsequent steps appear non-reversible.
In Plasmodium knowlesi, Plasmodium vivax and Plasmodium falciparum, the principal invasion pathway uses a merozoite protein expressed from a homologous family of erythrocyte binding proteins (the EBP family) [Adams et al., Proc. Natl. Acad. Sci. USA 89, 7085-9, (1992)]. The EBP proteins are transmembrane proteins, characterized by two conserved cysteine-rich domains, and are expressed in the micronemes (an apical organelle thought to play a crucial role in host cell invasion) of invasive merozoites. The EBP family includes the Plasmodium vivax/P. knowlesi Duffy binding protein (DBP) family and the P. falciparum erythrocyte binding antigen-175 (EBA-175). In particular, The P. vivax and P. knowlesi Duffy antigen binding proteins bind to Duffy blood group antigens of human erythrocytes, and the P. falciparum erythrocyte binding antigen-175 (EBA-175) binds to sialic acid-dependent determinants on glycophorin A of human erythrocytes.
The EBP family has a common gene structure, including 4-5 exons encoding separate functional domains (signal, ligand, transmembrane cytoplasmic domains) and conserved codon usage at exon-intron boundaries. The genes are typically present in single-copy with the exception being P. knowlesi which has 3-5 genes. EBPs are expressed as transmembrane proteins in late schizont development and are located in the merozoite microneme. The encoded proteins have two characteristic conserved cysteine-rich domains [an amino cysteine-rich (region II) and carboxyl cysteine-rich (region VI) domain] separated by a hydrophilic region.
The cysteine-rich domains have a highly conserved core structure among Plasmodium species. Domains similar to the amino cysteine-rich domain of the DBP and the EBA-175 have been described in the P. falciparum var gene family. Var genes code for variant proteins (PfEMP-1) which are expressed on the surface of infected erythrocytes and mediate cytoadherence. The high conservation of the cysteine-rich domains suggests that they play a crucial role in host cell receptor recognition. This hypothesis was experimentally verified by showing that the amino cysteine-rich domain of the EBPs is the principal adhesion domain involved in receptor binding [Adams et al., Proc. Natl. Acad. Sci. USA 89, 7085-9, 1992; Chitnis et al., J. Exp. Med. 180, 497-506, 1994; Sim et al., Science 264, 1941-1944, 1994]. In particular, variation in the EBP region II has been correlated with differences in receptor recognition [Adams et al., Proc. Natl. Acad. Sci. USA 89, 7085-9, 1992; Chitnis et al., J. Exp. Med. 180, 497-506, 1994]. Region VI is a smaller, carboxyl domain that has no identified function, but is more highly conserved than region II.
Plasmodium falciparum merozoites are primarily dependent on a glycophorin A pathway for invasion of erythrocytes, but have the ability to develop or utilize an alternate invasion pathway when glycophorin A is absent [Pasvol et al., Nature 297, 64-6, 1982; Hadley et al., J. Clin. Invest. 80, 1190-3, 1987; Dolan et al., Acta Leiden 60, 93-9, 1991; Dolan et al., J. Clin. Invest. 86, 618-24, 1990; Dolan et al., Mol. Biochem. Parasitol. 64, 55-63, 1994; Perkins et al., Mol. Biochem. Parasitol. 27, 23-34, 1988]. The MAEBL protein disclosed herein represents a ligand of an alternate pathway of invasion for Plasmodium parasites.
MAEBL is a single-copy gene with a multi-exon structure like EBP, including conserved intron/exon boundaries. The carboxyl end of MAEBL is homologous to EBPs and the carboxyl cys-rich domain has approximately 50% identity with EBPs. A 50 amino acid tandem repeat spans the region between the cysteine domains, maintaining the equivalent spacing between the cysteine domains as in other EBPs. The putative ligand domain of MAEBL has identity to AMA-1 instead of the consensus DBL motif of EBPs. The MAEBL AMA-1-like regions are present as a tandem duplication (YM-M1, YM-M2) FIG. 2c analogous to the tandem DBL duplication found in P. falciparum EBL (EBA-175). (See FIG. 2d) The second copy of the AMA-1-like domain has greater identity to the AMA-1 consensus sequence. Differences between MAEBL and AMA-1 occur mostly in the loop regions between predicted disulfide bridges and includes an alternatively-spliced cryptic intron in YM-M2. Unlike AMA-1, the primary nucleotide sequences of the MAEBL AMA-1-like domains are poorly conserved, indicating a possible ancient and evolving gene origin parallel to the current AMA-1. MAEBL still retains amino acid sequence similarity to the part of the EBP molecule that connects to the merozoite (and likely transmits an internal signal associated with receptor recognition) thus indicating its equivalent but alternate role in receptor recognition.